1. Platelets can enhance vascular permeability
by Nathalie Cloutier, Alexandre Paré, Richard W. Farndale, H. Ralph Schumacher, Peter A. Nigrovic, Steve Lacroix, and Eric Boilard
Blood
Volume 120(6):
August 9, 2012
©2012 by American Society of Hematology

2. Gaps are present between endothelial cells in the synovium vasculature from patients with RA. Synovium from RA patients was scrutinized by electron microscopy.
Gaps are present between endothelial cells in the synovium vasculature from patients with RA. Synovium from RA patients was scrutinized by electron microscopy. Black arrows indicate one gap between endothelial cells. E indicates endothelial cells; PL, platelets; L, lymphocytes; and RBC, red blood cells. Original magnification ×
Nathalie Cloutier et al. Blood 2012;120:
©2012 by American Society of Hematology

3. Presence of vasculature permeability during autoimmune arthritis in vivo.
Presence of vasculature permeability during autoimmune arthritis in vivo. (A) Nonarthritic control (left) and arthritic (right) mice (day 7 after K/BxN serum injection) were injected intravenously with 0.45-, 0.84-, 3.2-, or 10.2-μm-diameter microspheres and visualized 5 minutes later using a Xenogen IVIS in vivo imaging system. Control and arthritic mice received the same concentration of fluorescent microspheres. Radiant efficiency quantifications in the ankle joints for control and arthritic mice for all microsphere sizes are presented at right. (B-C) In vivo 2-photon imaging of the vasculature permeability in the arthritic ankle joint. LysM-eGFP arthritic mice injected intravenously with dextran-fluorescein and 0.45 μm Nile-Red-conjugated microspheres, and ankle joints were imaged to visualize the microsphere egress from the blood circulation and accumulation in the subendothelial collagen-rich matrix. (B) Two-photon images from time-lapse recordings taken at 0, 120, and 444 seconds demonstrating a microsphere leaving the blood circulation independently of transportation by a neutrophil or a monocyte. (C) Representative image evidencing the accumulation of microspheres outside the vasculature in an arthritic joint. Red represents microspheres; green, blood vessels; cyan blue, leukocytes; and Indigo blue, collagen (second-harmonic generation). Scale bar represents 25 μm (panel B), and 50 μm (panel C).
Nathalie Cloutier et al. Blood 2012;120:
©2012 by American Society of Hematology

4. The presence of gaps in the inflamed arthritic joint vasculature depends on platelets.
The presence of gaps in the inflamed arthritic joint vasculature depends on platelets. (A) The platelet-depleting antibody or the isotype control antibody was injected to mice on day 0 and day 3. Arthritis was induced by injection of 75 μL of K/BxN serum on day 0 and day 2. On day 4, the 0.45-μm microspheres were intravenously injected in platelet (PLT)–depleted mice and control mice (with PLT), and the fluorescence was measured in ankle joints 5 minutes later. Nonarthritic mice were used as controls. Representative results are presented. ***P < (B) The signs of arthritis were monitored daily and presented as mean ± SEM. Arrow indicates parenteral administration of platelet-depleting antibody or isotypic control; and arrowheads, K/BxN serum administration. (C) Antibody injection, arthritis induction, microsphere injection, fluorescence measurements, controls, and results presentation were performed as in panel A. Mouse recombinant IL-1β was injected on days 0, 1, and 2 in mice receiving the platelet-depleting antibody. ***P = (top). ***P = (bottom). (D) The signs of arthritis were monitored as in panel B. Arrow indicates parenteral administration of platelet-depleting antibody or isotypic control; arrowheads, K/BxN serum administration; and double arrow, IL-1β administration.
Nathalie Cloutier et al. Blood 2012;120:
©2012 by American Society of Hematology

5. Serotonin is present in the SF of patients with RA and can promote the gap formation in joint vasculature.
Serotonin is present in the SF of patients with RA and can promote the gap formation in joint vasculature. (A) Serotonin concentrations were measured in SF of RA and OA patients using an ELISA serotonin kit (n = 27). **P = (B) Serotonin suffices to promote prompt formation of gaps in joints. Diluent (PBS) or serotonin (20 mg/kg) was injected intravenously in mice before administration of 0.45-μm-diameter fluorescent microspheres. The fluorescence was portrayed using an in vivo imaging system 45 minutes after the microsphere injection (left). The radiant efficiency quantifications in the ankle joints for control (diluent) and serotonin injections are presented on the right. ***P < .0001; *P = .024.
Nathalie Cloutier et al. Blood 2012;120:
©2012 by American Society of Hematology

6. Mice deficient in platelet-derived serotonin have reduced gap formation.
Mice deficient in platelet-derived serotonin have reduced gap formation. (A) Serotonin concentrations were measured in whole blood from mast cell-deficient mice (KitW-sh), neutrophil-depleted mice, platelet-depleted mice, and their corresponding control mice using an ELISA serotonin kit. (B) Serotonin concentration was measured in platelet isolated from WT and Slc6a4−/− mice. (C) WT and Slc6a4−/− arthritic mice were injected intravenously with 0.45-μm-diameter fluorescent microsphere, and the fluorescence was visualized 5 minutes later using an in vivo imaging system. Control mice (WT nonarthritic) and WT and Slc6a4−/− arthritic mice all received the same concentration of fluorescent microspheres. The radiant efficiency quantifications in the ankle joints for all mice are presented, and representative results are presented at right. ***P = (D) Mice (17/group) sufficient and deficient in SERT expression were injected with 125 μL K/BxN serum at day 0 and day 2 and the signs of arthritis monitored daily. The mouse arthritis experiment is presented as mean ± SEM. P < .01. (E) SERT pharmacologic inhibition was performed in C57BL/6J mice by treating the mice with fluoxetine for 21 days before administration of K/BxN serum. Arthritic (treated or not with fluoxetine) and control (nonarthritic) mice were injected intravenously with 0.45-μm-diameter fluorescent microsphere, and the fluorescence was visualized 5 minutes later using an in vivo imaging system. The radiant efficiency quantifications in the ankle joints for all mice are presented, and representative results are presented at right. **P = (F) Serotonin concentrations in platelets isolated from mice treated or not with fluoxetine were measured by ELISA.
Nathalie Cloutier et al. Blood 2012;120:
©2012 by American Society of Hematology

7. GPVI activation induces the formation of gaps in joints via release of serotonin.
GPVI activation induces the formation of gaps in joints via release of serotonin. Nonarthritic mice (treated or not with fluoxetine for 21 days) were injected intravenously with CRP (40 μg/kg) or its diluent PBS. Thirty minutes after the CRP injection, the 0.45-μm fluorescent microspheres were injected. Two minutes after the microsphere injections, the fluorescence in ankle joints was evaluated using an in vivo imaging system. The radiant efficiency quantifications in the ankle joints for all mice and representative results are presented. ***P < .0001; **P =
Nathalie Cloutier et al. Blood 2012;120:
©2012 by American Society of Hematology

8. Proposed pathway for the formation of gaps and amplification of the vasculature permeability by platelets during arthritis.
Proposed pathway for the formation of gaps and amplification of the vasculature permeability by platelets during arthritis. Top panel: Gaps between endothelial cells in arthritic joints are formed. The GPVI-expressing platelets are activated by the newly exposed subendothelial matrix rich in GPVI ligands, such as collagen and laminin. Bottom panel: Stimulated platelets produce copious amounts of IL-1–rich microparticles and release serotonin. Platelet-derived serotonin promotes the production of additional gaps; thus, more platelets can be activated. Serotonin at the site of inflammation promotes the vasculature leakage during disease. Note that the precise anatomic location of platelet activation and the route by which microparticles enter the joint remain speculative. ▴ represents serotonin; and ○, platelet microparticle. Illustration courtesy of Steve Moskowitz, Advanced Medical Graphics.
Nathalie Cloutier et al. Blood 2012;120:
©2012 by American Society of Hematology